Electrophilic Modification of PKM2 by 4-Hydroxynonenal and 4

Dec 15, 2016 - Electrophilic Modification of PKM2 by 4‑Hydroxynonenal and. 4‑Oxononenal Results in Protein Cross-Linking and Kinase Inhibition. Je...
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Electrophilic Modification of PKM2 by 4-Hydroxynonenal and 4Oxononenal Results in Protein Cross-Linking and Kinase Inhibition Jeannie M Camarillo, Jody Christine Ullery, Kristie L. Rose, and Lawrence J. Marnett Chem. Res. Toxicol., Just Accepted Manuscript • DOI: 10.1021/acs.chemrestox.6b00374 • Publication Date (Web): 15 Dec 2016 Downloaded from http://pubs.acs.org on December 19, 2016

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Electrophilic modification of PKM2 by 4-hydroxynonenal and 4-oxononenal results in protein cross-linking and kinase inhibition Jeannie M. Camarillo†#, Jody C. Ullery†#, Kristie L. Rose‡, Lawrence J. Marnett†¥*

† ‡ ¥

Department of Biochemistry

Mass Spectrometry Research Center, Vanderbilt University

A.B. Hancock Jr. Memorial Laboratory for Cancer Research, Department of Biochemistry,

Chemistry, and Pharmacology, Vanderbilt Institute of Chemical Biology, Center in Molecular Toxicology, Vanderbilt-Ingram Cancer Center Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146

#

These authors contributed equally to this work.

*To whom correspondence should be addressed: Address: Department of Biochemistry, Vanderbilt University, School of Medicine, 23rd Avenue South & Pierce, Room 850 RRB, Nashville, TN 37232-0146 Phone: 615-343-7329 Fax: 615-343-7534 E-mail: [email protected] Graphical Abstract

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Abstract Rapidly proliferating cells require increased metabolism to allow for the production of nucleic acids, amino acids, and lipids. Pyruvate kinase catalyzes the final step in the glycolysis pathway and different isoforms display vastly different catalytic efficiencies. The M2 isoform of pyruvate kinase (PKM2) is highly expressed in cancer cells and contributes to aerobic glycolysis in what is commonly referred to as the Warburg effect. Here, we show that PKM2 is covalently modified by the lipid electrophiles 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE). HNE and ONE modify multiple sites on PKM2 in vitro, including Cys424 and His439, which play a role in protein-protein interactions and fructose 1,6-bis-phosphate binding, respectively. Modification of these sites results in a dose-dependent decrease in enzymatic activity. In addition, high concentrations of electrophile, most notably in the case of ONE, result in substantial proteinprotein cross-linking in vitro and in cells. Exposure of RKO cells to electrophiles results in modification of monomeric PKM2 in a dose-dependent manner. There is a concomitant decrease in PKM2 activity in cells upon ONE exposure, but not HNE exposure. Together, our data suggest that modification of PKM2 by certain electrophiles results in kinase inactivation.

Introduction Early studies by Otto Warburg identified aerobic glycolysis as the main mechanism by which cancer cells generate ATP. Elevated glucose uptake, increased glycolysis, and high lactate production, even in the presence of oxygen, allow for more rapid cell growth and tumorigenesis.1 Malignant tumors can have glycolytic rates up to 200 times that of normal tissues, and the switch from oxidative phosphorylation to aerobic glycolysis is coupled with a switch in isoforms of the glycolytic enzyme, pyruvate kinase.2

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Pyruvate kinase catalyzes the final reaction in glycolysis, the transfer of a phosphate group from phosphoenolpyruvate to ADP, to yield one molecule of pyruvate and one molecule of ATP. Expression of pyruvate kinase isoform M2 (PKM2) occurs during fetal development, while the M1 isoform predominates throughout the majority of adult tissues. During tumorigenesis, PKM2 becomes the predominant isoform.2 The lower enzymatic activity of PKM2 relative to that of PKM1 results in increased glycolytic intermediates that can be used for different biosynthetic pathways, thereby contributing to more rapid cell growth.3 PKM2 has also been identified as a regulator of transcription. Translocation of PKM2 to the nucleus allows for coactivation of β-catenin, induction of c-Myc expression, and upregulation of genes which contribute to the Warburg effect.4,5 Regulation of PKM2 activity has been an active area of research for a number of years. PKM2 glycolytic and transcriptional activity is modulated by a number of post-translational modifications, multimerization, and allosteric interactions.6 Fructose 1,6-bis-phosphate allosterically binds PKM2, promoting homotetramer formation and increasing glycolytic activity.2 Acetylation of PKM2 at K433 inhibits this allosteric interaction, resulting in formation of PKM2 dimers that can translocate to the nucleus and transcriptionally regulate genes involved in cell cycle progression and glycolysis.7 These two different functions of PKM2 are thought to further contribute to tumorigenesis. Lipid electrophiles are generated during periods of oxidative stress and are associated with a multitude of diseases, including cancer. 4-Hydroxy-2-nonenal (HNE) and 4-oxo-2nonenal (ONE) readily react with Cys, His, and Lys residues to form Michael adducts, whereas ONE displays unique reactivity toward Lys residues to produce the Schiff base and the 4-

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ketoamide adducts.8,9 Modification of proteins has typically been shown to result in loss of protein function and to promote dysregulation of multiple cell signaling pathways. Recent work has used alkyne analogues of HNE and ONE, aHNE and aONE, respectively, to selectively isolate adducted proteins via click chemistry for identification by mass spectrometry. Using this approach, Codreanu et al.10 identified >1000 proteins as targets of aHNE or aONE modification. Interestingly, these targets displayed a range of sensitivities to each electrophile. PKM2 was shown to be modified by both aHNE and aONE, and was heavily modified at low concentrations of both electrophiles, revealing it as a preferential target of adduction. Here, we map the sites of PKM2 modification by HNE and ONE and show that these modifications result in PKM2 cross-linking in vitro and in cells. Additionally, cross-linking by ONE produces a fully functioning PKM2 dimer in vitro. In cells, PKM2 is adducted by aHNE and aONE at 10 µM (Figure 2B). These data suggest that ONE-mediated cross-linking of PKM2 may contribute to altered activity in the cell.

HNE and ONE inhibit kinase activity in recombinant PKM2 protein. Due to the ability of HNE and ONE to cross-link PKM2 monomers, we assessed the impact of electrophile treatment and subsequent protein adduction on PKM2 kinase activity. Recombinant PKM2 protein was incubated with increasing concentrations of HNE or ONE that were known to contribute to dimer, trimer, and tetramer formation, and PKM2 kinase activity was assessed by a lactate dehydrogenase-coupled assay to measure the oxidation of NADH to NAD+.12 In vitro adduction of PKM2 with HNE resulted in a significant decrease in PKM2 kinase activity at HNE concentrations greater than 40 µM (Figure 3A). These concentrations only contributed to a low percentage of cross-linked dimer at similar concentrations. These data suggest that PKM2 activity decreases with increasing HNE adduction.

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Conversely, treatment with ONE resulted in a significant decrease in PKM2 kinase activity at low electrophile concentration. When recombinant PKM2 protein was treated with increasing concentrations of ONE in vitro, activity assays showed >50% reduction in PKM2 kinase activity at 10 µM ONE, with complete inhibition at higher concentrations (Figure 3B). Interestingly, there was little difference in activity at low concentrations of ONE, which were shown to crosslink PKM2 monomers to dimers. It is possible that ONE-induced cross-linking of PKM2 alone does not result in inhibition of activity.

PKM2 is adducted by electrophiles in RKO cells. To verify modification of PKM2 in RKO cells, cells were treated with aHNE and aONE to enable selective tagging and isolation of adducted proteins by click chemistry.11 RKO cells were treated with aHNE or aONE for 1 h, lysed, and the protein extract reduced and subjected to click chemistry with a UV-cleavable N3-biotin. Photoelution from streptavidin beads allowed for selective elution of adducted proteins. Western blot analysis of eluates revealed that PKM2 was modified by both aHNE (Figure 4A) and aONE (Figure 4B) in a dose-dependent manner. Only PKM2 monomers were recovered at the concentrations of aHNE and aONE that we investigated, which suggests that multimerization of PKM2 only occurs at higher concentrations.

PKM2 is cross-linked by aONE in RKO cells. While we did not observe multimers of PKM2 in the click elution (Figure 4), we wanted to definitively assess if PKM2 can be cross-linked by electrophiles in the cell. Following treatment of RKO cells with increasing concentrations of HNE or ONE, we performed western blot analysis to assess if dimers of PKM2 were visible. Interestingly, high concentrations of ONE (>100 µM) resulted in the appearance of PKM2

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dimers in whole cell lysates (Figure 5). Dimers were not observed in lysates that had been treated with HNE (data not shown). These data show that cross-linking of PKM2 can occur in living cells; however high levels of exogenous ONE produce only low levels of cross-linking.

ONE inhibits kinase activity in cellular PKM2. To begin to elucidate the biological impact of electrophile modification of PKM2, kinase assays were performed on RKO cell lysates. RKO cells were treated with increasing concentrations of HNE and ONE, and lysates were reduced with NaBH4. In cells treated with HNE, no differences were observed in PKM2 activity at any of the concentrations we tested (Figure 6A). Interestingly, cells treated with ONE displayed a significant decrease in PKM2 activity at high concentrations (Figure 6B). It is notable that this altered activity of PKM2 in response to ONE correlates with the cross-linking that is observed at high cellular ONE concentrations (Figure 5).

Discussion While the chemistry of lipid peroxidation and subsequent protein adduction has been extensively investigated, the number of protein targets and the potential biological implications are only recently being elucidated. HNE was identified prior to ONE, thus it has been the most highly studied product of autoxidation of polyunsaturated fatty acids. Upon the discovery of ONE, studies began to focus on the increased reactivity of ONE relative to HNE.17 These studies led to the identification of a number of new ONE-derived modifications, including cross-links formed between Cys or His Michael adducts and Schiff base-derived adducts on Lys residues. Previous work has investigated the protein targets of different lipid electrophiles in multiple cell types. While these large-scale proteomic data sets have provided insight into the

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signaling pathways that could be disrupted as a result of electrophile modification, the precise impact on protein function remains unknown for the majority of modified proteins. Our results have not only investigated the effect on kinase activity of a key glycolytic protein, but also provided insight into how its modification could influence cancer progression. Using HNE and ONE treatment in vitro, we have shown by mass spectrometry that PKM2 is adducted at Cys, His, and Lys residues in the form of Michael adducts, as well as a Lys residue in the form of the 4-ketoamide. Electrophile treatment, most notably with ONE, results in cross-linking of PKM2 monomers. ONE cross-links can occur between Cys/Lys and His/Lys,18,19 and the susceptibility of these residues to cross-linking has been exemplified with other α,βunsaturated carbonyls, such as cis-2-butene-1,4-dial.20 In all these examples, the cross-link is an N-substituted pyrrole, but observation of this structure was only on individual amino acids or single tryptic peptides. Other cross-link structures, such as a Lys-Lys cross-link, have also been proposed to occur with HNE and ONE.21,22 Technology limitations make it difficult to determine inter-peptide cross-links by mass spectrometry. While SDS-PAGE and Coomassie blue staining show that treatment with either HNE or ONE results in cross-linking between PKM2 monomers in vitro, manual investigation of the proteomic data did not yield any potential peptide crosslinks. Additionally, these proteomic data show adducts occurring on digested monomers, but not on multimers, suggesting that the majority of adducts on multimers may be within cross-links. PKM2 activity in vitro is significantly decreased in response to both HNE and ONE. The loss of activity correlates with the increase in cross-linking, and it is possible that this loss of activity is the direct result of cross-linking. Additional hypotheses can also explain the loss of activity. Adduction of specific sites could contribute to the disruption of substrate binding, thereby reducing catalytic activity independent of cross-linking. While none of the sites of

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adduction we found are known to directly interact with substrate binding, it is possible that adduction could disrupt the local structure to alter binding. Consistent with observations in vitro, our results in RKO cells show that PKM2 is highly susceptible to electrophile adduction. Click chemistry and selective isolation of adducted proteins shows measurable adduction of PKM2 monomers at concentrations as low as 5-10 µM. Increased PKM2 adduction in cells by aONE is likely due to the higher reactivity of aONE versus aHNE. High ONE levels resulted in formation of cross-linked PKM2 dimers and contributed to decreased kinase activity. While the levels of ONE required to induce cross-link formation and loss of activity in cells are much higher than in vitro, these results are likely due the additional protein targets in cells and the possibility that not all the exogenous electrophile penetrates the cell. ONE is a more potent cross-linker than HNE, and the cross-links generated are thought to be chemically stable.18,23 The lack of cross-linking and altered activity of PKM2 in response to HNE suggests different residues of modification in cells between the two electrophiles. In other words, HNE may target sites that do not play a role in substrate, allosteric, or protein binding, thereby making these adducts benign to PKM2 activity. ONE appears to disrupt PKM2 activity in cells, so the modified residues may play a more significant role in catalysis or protein binding. Ultimately, ONE has a more potent inhibition of PKM2 activity, and we propose that inhibition of PKM2 glycolytic activity is due to the cross-linking of active dimers to tetramers (Figure 7). Methods to determine sites of cross-links is an area of further development and investigation.

Funding Information This work was supported by the NCI F31 CA192861 (J.M.C.) and R37 CA087819 (L.J.M.). Acknowledgements

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The authors would like to thank Dr. Carol Rouzer for critical review of the manuscript. Supporting Information MS2 spectra of adducted peptides. This material is available free of charge via the Internet at http://pubs.acs.org. Abbreviations HNE – 4-Hydroxy-2-nonenal; ONE – 4-Oxo-2-nonenal; DMSO – Dimethyl Sulfoxide; RT – Room Temperature; aHNE – Alkynyl 4-Hydroxy-2-nonenal; aONE – Alkynyl 4-Oxo-2-nonenal; PKM2 – Pyruvate Kinase Isoform M2; TBST – Tris-Buffered Saline with Tween-20; DPBS – Dulbecco’s Phosphate Buffered Saline; FBS – Fetal Bovine Serum.

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References (1) Warburg, O. (1956) On the origin of cancer cells. Science. 123, 309-314. (2) Mazurek, S., Boschek, C. B., Hugo, F. and Eigenbrodt, E. (2005) Pyruvate kinase type M2 and its role in tumor growth and spreading. Seminars in cancer biology. 15, 300-308. (3) Chaneton, B., Hillmann, P., Zheng, L., Martin, A. C. L., Maddocks, O. D. K., Chokkathukalam, A., Coyle, J. E., Jankevics, A., Holding, F. P., Vousden, K. H., Frezza, C., O/'Reilly, M. and Gottlieb, E. (2012) Serine is a natural ligand and allosteric activator of pyruvate kinase M2. Nature. 491, 458-462. (4) Yang, W., Xia, Y., Ji, H., Zheng, Y., Liang, J., Huang, W., Gao, X., Aldape, K. and Lu, Z. (2011) Nuclear PKM2 regulates β-catenin transactivation upon EGFR activation. Nature. 480, 118-122. (5) Yang, W., Zheng, Y., Xia, Y., Ji, H., Chen, X., Guo, F., Lyssiotis, C. A., Aldape, K., Cantley, L. C. and Lu, Z. (2012) ERK1/2-dependent phosphorylation and nuclear translocation of PKM2 promotes the Warburg effect. Nat Cell Biol. 14, 1295-1304. (6) Wong, N., Ojo, D., Yan, J. and Tang, D. (2015) PKM2 contributes to cancer metabolism. Cancer Letters. 356, 184-191. (7) Lv, L., Xu, Y. P., Zhao, D., Li, F. L., Wang, W., Sasaki, N., Jiang, Y., Zhou, X., Li, T. T., Guan, K. L., Lei, Q. Y. and Xiong, Y. (2013) Mitogenic and oncogenic stimulation of K433 acetylation promotes PKM2 protein kinase activity and nuclear localization. Mol Cell. 52, 340-352. (8) Esterbauer, H., Schaur, R. J. and Zollner, H. (1991) Chemistry and biochemistry of 4hydroxynonenal, malonaldehyde and related aldehydes. Free radical biology & medicine. 11, 81-128. (9) Ullery, J. C. and Marnett, L. J. (2012) Protein modification by oxidized phospholipids and hydrolytically released lipid electrophiles: Investigating cellular responses. Biochim Biophys Acta. 1818, 2424-2435. (10) Codreanu, S. G., Ullery, J. C., Zhu, J., Tallman, K. A., Beavers, W. N., Porter, N. A., Marnett, L. J., Zhang, B. and Liebler, D. C. (2014) Alkylation damage by lipid electrophiles targets functional protein systems. Mol Cell Proteomics. 13, 849-859. (11) Vila, A., Tallman, K. A., Jacobs, A. T., Liebler, D. C., Porter, N. A. and Marnett, L. J. (2008) Identification of protein targets of 4-hydroxynonenal using click chemistry for ex vivo biotinylation of azido and alkynyl derivatives. Chem Res Toxicol. 21, 432-444. (12) Christofk, H. R., Vander Heiden, M. G., Harris, M. H., Ramanathan, A., Gerszten, R. E., Wei, R., Fleming, M. D., Schreiber, S. L. and Cantley, L. C. (2008) The M2 splice isoform of pyruvate kinase is important for cancer metabolism and tumour growth. Nature. 452, 230-233. (13) Camarillo, J. M., Rose, K. L., Galligan, J. J., Xu, S. and Marnett, L. J. (2016) Covalent Modification of CDK2 by 4-Hydroxynonenal as a Mechanism of Inhibition of Cell Cycle Progression. Chem Res Toxicol. 29, 323-332. (14) Uchida, K. and Stadtman, E. R. (1992) Modification of histidine residues in proteins by reaction with 4-hydroxynonenal. Proc Natl Acad Sci U S A. 89, 4544-4548. (15) Gui, D. Y., Lewis, C. A. and Vander Heiden, M. G. (2013) Allosteric Regulation of PKM2 Allows Cellular Adaptation to Different Physiological States. Science Signaling. 6, pe7-pe7.

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(16)

(17)

(18)

(19)

(20)

(21) (22) (23)

Stewart, B. J., Doorn, J. A. and Petersen, D. R. (2007) Residue-Specific Adduction of Tubulin by 4-Hydroxynonenal and 4-Oxononenal Causes Cross-Linking and Inhibits Polymerization. Chemical Research in Toxicology. 20, 1111-1119. Doorn, J. A. and Petersen, D. R. (2002) Covalent modification of amino acid nucleophiles by the lipid peroxidation products 4-hydroxy-2-nonenal and 4-oxo-2nonenal. Chem Res Toxicol. 15, 1445-1450. Aluise, C. D., Camarillo, J. M., Shimozu, Y., Galligan, J. J., Rose, K. L., Tallman, K. A. and Marnett, L. J. (2015) Site-specific, intramolecular cross-linking of Pin1 active site residues by the lipid electrophile 4-oxo-2-nonenal. Chem Res Toxicol. 28, 817-827. Oe, T., Arora, J. S., Lee, S. H. and Blair, I. A. (2003) A novel lipid hydroperoxidederived cyclic covalent modification to histone H4. The Journal of biological chemistry. 278, 42098-42105. Chen, L.-J., Hecht, S. S. and Peterson, L. A. (1997) Characterization of Amino Acid and Glutathione Adducts of cis-2-Butene-1,4-dial, a Reactive Metabolite of Furan. Chemical Research in Toxicology. 10, 866-874. Sayre, L. M., Lin, D., Yuan, Q., Zhu, X. and Tang, X. (2006) Protein adducts generated from products of lipid oxidation: focus on HNE and one. Drug Metab Rev. 38, 651-675. Nadkarni, D. V. and Sayre, L. M. (1995) Structural definition of early lysine and histidine adduction chemistry of 4-hydroxynonenal. Chem Res Toxicol. 8, 284-291. Oe, T., Lee, S. H., Silva Elipe, M. V., Arison, B. H. and Blair, I. A. (2003) A novel lipid hydroperoxide-derived modification to arginine. Chem Res Toxicol. 16, 1598-1605.

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Table Legend Table 1. PKM2 is modified at multiple sites by HNE and ONE. Recombinant PKM2 was modified in vitro with increasing concentrations of HNE or ONE, adducts were reduced, and proteins resolved by SDS-PAGE prior to in-gel digestion and analysis by LC-MS/MS. Peptide

Mass Observed Theoretical Charge Error m/z m/z (ppm)

Residue

Modification

Electrophile

NTGIIC*TIGPASR

730.9112 730.9107

2

0.68

Cys49

Michael Adduct

HNE and ONE

IENH*EGVR

556.3110 556.3089

2

3.77

His274

Michael Adduct

HNE and ONE

CC*SGAIIVLTK

661.8759 661.8747

2

1.81

Cys424

Michael Adduct

HNE and ONE

SAH*QVAR

463.7764 463.7745

2

4.10

His439

Michael Adduct

HNE and ONE

NIK*IISK

486.3305 486.3286

2

3.91

Lys256

Michael Adduct

HNE

VLGEK*GK

443.7864 443.7840

2

5.41

Lys261

Ketoamide

ONE

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Figure Legend Figure 1. Structures of electrophiles and adducts. A) Structures of electrophiles used in these studies, HNE and ONE, and the alkyne analogues, aHNE and aONE. B) Structures of adducts found on PKM2. Cys and His adducts were found with in both HNE and ONE. Michael adducts of HNE were found on Lys while ONE formed the 4-ketoamide adduct on Lys. Figure 2. PKM2 cross-links occur upon exposure to HNE and ONE. Recombinant PKM2 was modified in vitro with increasing concentrations of A) HNE or B) ONE for 1 h at 37°C. Staining of SDS-PAGE with Coomassie blue shows cross-linking of PKM2 monomers with increasing concentrations of electrophile. Figure 3. Cross-linking inhibits kinase activity in recombinant PKM2 protein. Recombinant PKM2 was modified in vitro with increasing concentrations of A) HNE or B) ONE for 1 h at 37°C. PKM2 activity was assessed indirectly by the lactate dehydrogenase-coupled enzyme assay. Data represent an n=3 ± SD, *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA. Figure 4. PKM2 is adducted by electrophiles in RKO cells. RKO cells were treated with increasing concentrations of aHNE or aONE for 1 h. Modified proteins were conjugated with UV-cleavable N3-biotin via click chemistry, bound to streptavidin beads, and eluted under 365 nm UV light. Western blot analysis of UV eluates and whole cell lysate (Input, 0.1%) showed a dose-dependent increase in modification of PKM2 by both electrophiles. Figure 5. PKM2 is cross-linked by aONE in RKO cells. RKO cells were treated with increasing concentrations of ONE for 1 h. After reduction of lysates with NaBH4, proteins were separated by SDS-PAGE and PKM2 levels were assessed with western blot. High concentrations of ONE resulted in formation of PKM2 cross-linked dimers. Figure 6. Oligomer formation inhibits kinase activity in cellular PKM2. RKO cells were treated with increasing concentrations of HNE or ONE for 1 h, lysed, reduced with NaBH4, and subjected to in vitro PKM2 kinase assays. Data represent the mean ± SD (n = 3), *P < 0.05, **P < 0.01, ***P < 0.001 by ANOVA. Figure 7. Proposed mechanism of PKM2 inhibition. Under normal conditions, PKM2 monomoer form tetramers to convert PEP to pyruvate. In vitro and in cells, ONE forms cross-links between PKM2 monomers. Dimer formation allows for catalytic activity while formation of tetramers contributes to enzyme inactivation.

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Figures Figure 1 A)

B)

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Figure 2 A) 0

50

100

HNE (µM)

200 150 Dimer 100

Monomer 50

B) 0

200 150

0.3125 0.625 1.25

2.5

5

10

20

40

ONE (µM)

Tetramer Dimer

100 50

Monomer

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Figure 3 A)

Relative PKM2 Activity

150

100

*

***

50

***

0 0

1

5

10

20

40

75

100

HNE (µM)

B) 125

Relative PKM2 Activity

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

100 75 50

***

25

*** ***

0 0

1

2.5

5

7.5

10

25

50

ONE (µM)

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Chemical Research in Toxicology

Figure 4 A)

B)

ACS Paragon Plus Environment

Chemical Research in Toxicology

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Figure 5

ACS Paragon Plus Environment

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Figure 6 A)

Relative PKM2 Activity

150

100

50

0 0

5

20

40

75

100

Concentration (µM)

B) 150

Relative PKM2 Activity

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Chemical Research in Toxicology

** 100

* ***

50

0 0

5

20

40

75

100

Concentration (µM)

ACS Paragon Plus Environment

Chemical Research in Toxicology

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Figure 7

ACS Paragon Plus Environment

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